Toward the end of the last century, the remarkable conjunction of the discoveries of mammalian cloning and human embryonic stem (ES) cells almost immediately gave rise to speculation that the fusion of these technologies might be used to generate customized pluripotent stem cell lines from individual patients (Wilmut (1998)). ES cells have the unique potential to differentiate into cell types of the three germ layers and the capacity of unlimited self renewal (Smith, 2001). Such cell lines could therefore help to overcome the formidable hurdle of immune rejection of transplants derived from allogeneic ES cells, and could also provide laboratory models for the study of complex human diseases. Proof of concept for this approach in the mouse was not long in coming (Munsie et al. (2000)), but ethical and practical barriers have slowed extension of somatic cell nuclear transfer to the human.
Although it was obvious that reprogramming of somatic cells by cloning or cell fusion (Tada et al. (1997)) must involve gene-factors present specifically in the egg or in pluripotent stem cells, both processes seemed highly complex, and the list of factors involved was potentially quite long. Yamanaka's landmark study (Takahashi& Yamanaka (2006)) was striking because it reduced the reprogramming process to the action of a few genes. Soon, the same group, and several others, showed that induced pluripotent stem (iPS) cells produced by modifications of the original method had identical properties to those of pluripotent stem cell lines derived from embryos (Okita et al. (2007) and Wernig et al. (2007)). However, chimeric mice derived from these cell lines still developed tumors, the formation of which was linked causally to use of the c-myc gene in the reprogramming protocol.
During the last years various studies were reported aiming at deciphering the molecular mechanisms that promote the exclusive properties of ES cells. Cytokines such as leukemia inhibitory factor (LIF) and bone morphogenic protein (BMP) turned out to be sufficient to maintain pluripotency of ES cells. Moreover, internal regulatory pathways consisting of a self-organized transcription factor network appear to play an essential role in the regulation of early embryonic development (for review see Niwa, 2007). In this respect the transcription factors Oct4, Sox2 and Nanog seem to form a core network for the regulation of pluripotency (Boyer et al., 2005; Loh et al., 2006). In addition epigenetic processes such as bivalent chromatin domains and polycomb group proteins are fundamental for the maintenance of pluripotency (Bernstein et al., 2006; Boyer et al., 2006).
A major breakthrough in stem cell research was the recently reported successful reprogramming of murine fibroblasts to induced pluripotent stem (iPS) cells (Takahashi et al., 2006; Okita et al., 2007; Wernig et al., 2007; Maherali et al., 2007). It turned out that four factors, namely Oct4, Sox2, Klf4 and cMyc are particularly capable of establishing pluripotency in somatic cells. The same factors were shown to be able to reprogram human somatic cells to iPS cells (Takahashi et al., 2007; Lowry et al., 2008). More recently it has been reported that cMyc is dispensable for reprogramming (Park et al., 2007; Nakagawa et al., 2008; Wernig et al., 2008). Thomson and co-workers showed that reprogramming of human ES cells is possible by another combination of factors which is Oct4, Sox2, Nanog and LIN28, whereas Oct4 and Sox2 were indispensable for this process in comparison to the other two factors (Yu et al., 2007).
Oct4 is a POU-domain transcription factor (encoded by Pou5f1) that is expressed in all blastomers of early developing embryos and later during embryonic development it is restricted to the inner cell mass (ICM). It is down regulated in trophectodermal and primitive endodermal tissues (Nichols et al., 1998; Niwa et al., 2000). RNAi interference (RNAi) knock down of Pou5f1 in murine ES cells results in differentiation (Hay et al., 2004; Hough et al., 2006). At maturity, Oct4 expression is exclusive to developing germ cells (Pesce and Scholer, 2001).
Sox2 is a member of the SRY-related HMG box transcription factor family, and exhibits a similar expression pattern to Oct4 in early embryonic development. Sox2 interacts with Oct4 to regulate pluripotency and segregation to the first three lineages (Avilion et al., 2003). A knock down of Sox2 by RNAi causes differentiation to multiple lineages including trophectoderm (Ivanova et al., 2006). Interestingly, a composite POU-HMG DNA-binding site was found to be conserved in the regulatory region of many developmental genes. The regulation of these genes by the interaction of POU homeodomain and HMG factors is thought to be a fundamental mechanism for the regulation of expression of developmental genes (Dailey & Basilico, 2001). Oct4 and Sox2 act cooperatively to activate the expression of several pluripotency factors such as Fgf4, Utf1, Fbx15, Nanog (Boyer et al., 2005; Loh et al., 2006) (Kuroda et al., 2005; Nishimoto et al., 1999; Rodda et al., 2005; Tokuzawa et al., 2003; Yuan et al., 1995) and additionally regulate the expression of their own genes (Chew et al., 2005; Okumura-Nakanishi et al., 2005; Tomioka et al., 2002).
So far all studies investigating the influence of particular transcription factors, such as Sox2 and Oct4, on reprogramming of adult stem cells to iPS cells were based on genetic modification. However, random integration of transgene sequences into the genome can cause insertional mutagenesis (Glover et al., 2005; Okita et al., 2007) which limits later therapeutically use immensely. Further, the forced transgene expression known within the state of the art (e.g. DNA transfection or viral transduction) is elaborate and expensive.
Therefore, there is a need for a method for the generation of induced pluripotent cells which do not involve genetic modification.
The inventors of the present invention devised that induced pluripotent cells can be generated by transduction of a particular fusion protein into adult (stem) cells.